Abstract: :
Purpose: Human cornea stromal cells are reported to be difficultto transfect. We hypothesize that these cells are capable ofuptake of transfected DNA, but have one or more mechanisms forrepressing expression from DNA introduced into cells. Methods:Stromal cells derived from donor corneas were transfected withluciferase–expressing reporter plasmid DNA using severaltransfection reagents. Gene expression was measured by chemiluminescenceassay of luciferase. Corneal cells were compared to U2OS cells,human osteosarcoma cells that are "easily transfected". To quantitateDNA uptake into cells, 3H–labeled plasmid DNA was transfected.Repression of gene expression from cellular DNA has been correlatedwith formation of heterochromatin characterized by nucleosomeassociation and histone hypoacetylation. Micrococcal nucleasedigestion was used to determine the association of plasmid DNAwith nucleosomes. Treatment with trichostatin A (TSA), an inhibitorof histone deacetylases, was used to assess the effects of histoneacetylation on gene expression from plasmid DNA. Results: Corneastromal cells and U2OS cells take up equivalent amounts of transfectedDNA. The DNA taken into cells is present in the cytoplasm andthe nucleus, and that in the nucleus is both soluble and insoluble.The distribution of plasmid DNA into these subcellular fractionsis similar in corneal cells and U2OS cells. However, expressionof the DNA taken into corneal cells is negligible compared toexpression in U2OS cells. The DNA localized to the nucleus ofeach cell type is associated with nucleosomes. Expression oftransfected DNA in both cell types is enhanced by treatmentof cells with TSA, suggesting that in both cell types, histonespresent on the DNA are not hypoacetylated. However, the levelof expression achieved following TSA treatment of corneal cellsremains much lower than that in U2OS cells, indicating thatthere is an additional mechanism of repression in cornea cellsbeyond histone deacetylation.Conclusions: The inability to detectexpression of DNA transfected into corneal cells is not dueto a block in DNA uptake, but rather due to the repression ofintranuclear tranfected DNA by deacetylation of histones associatedwith the DNA as well as an additional repressive mechanism.